Question

A protein has a molecular mass of \(400 \mathrm{kDa}\) when measured by size- exclusion chromatography. When subjected to gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), the protein gives three bands with molecular masses of 180,160 , and 60 \(\mathrm{kDa}\). When electrophoresis is carried out in the presence of SDS and dithiothreitol, three bands again form, this time with molecular masses of 160, 90, and \(60 \mathrm{kDa}\). How many subunits does the protein have, and what is the molecular mass of each?

Short answer

The protein has 5 subunits: two 160 kDa, two 90 kDa, and one 60 kDa.

Step-by-step solution

Determine Initial Subunit Configuration

From the size-exclusion chromatography, the protein appears to be a single large unit with a molecular mass of \(400\ \mathrm{kDa}\). This suggests it may be composed of smaller subunits when fully separated by SDS-PAGE.

Analyze SDS-PAGE Results Without Dithiothreitol

In SDS-PAGE without \(\text{DTT}\), the protein separates into bands of \(180\ \mathrm{kDa}\), \(160\ \mathrm{kDa}\), and \(60\ \mathrm{kDa}\). This suggests the protein comprises three different types of subunits or aggregates.

Examine SDS-PAGE Results With Dithiothreitol

When \(\text{DTT}\) is added, which reduces disulfide bonds, the protein separates into \(160\ \mathrm{kDa}\), \(90\ \mathrm{kDa}\), and \(60\ \mathrm{kDa}\) bands. This indicates the breaking of disulfide-linked subunits.

Deduce Subunit Structure and Number

Comparing both cases, it appears the \(180\ \mathrm{kDa}\) band splits into \(90\ \mathrm{kDa}\) when reducing agent is present. Thus, \(180\ \mathrm{kDa}\) consists of two \(90\ \mathrm{kDa}\) subunits linked by disulfide bonds.Therefore, the protein contains:- Two \(160\ \mathrm{kDa}\) subunits- Two \(90\ \mathrm{kDa}\) subunits- One \(60\ \mathrm{kDa}\) subunit.

Key concepts

Molecular Mass Determination

Determining the molecular mass of a protein is crucial in understanding its structure and function. The molecular mass is assessed by techniques such as size-exclusion chromatography and SDS-PAGE, which we will explore further below. Size-exclusion chromatography estimates the mass based on the protein's elution volume, allowing us to infer whether the protein acts as a single entity or multiple components.
In the case of the exercise, the protein was found to have a mass of 400 kDa in size-exclusion chromatography. However, this value suggests that the protein might be assembled from smaller subunits, as concluded when using other techniques. The confirmation comes from additional analytical methods such as SDS-PAGE, which offers further insights into the subunit composition.

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used laboratory technique in determining protein molecular masses and their subunit compositions. SDS acts by coating protein molecules with a negative charge, which causes them to denature into linear forms and migrate towards the positive electrode during electrophoresis. This ensures that migration rates depend only on size, allowing separation based on molecular mass.

• This technique allows us to detect multiple components within a protein complex, as was seen in the original exercise with bands at 180, 160, and 60 kDa.
• The patterns of migration provide evidence of distinct subunits or aggregates, especially when treatments like reducing agents are added.

Understanding how SDS-PAGE works is critical, as it provides us with the evidence needed to infer protein structure and potential assembly from subunits.

Protein Subunits

Proteins can consist of multiple smaller units known as subunits. These subunits can be identical or different, forming complex structures through non-covalent interactions and often disulfide bonds. Methods like SDS-PAGE help to reveal these subunits by separating them based on size under denaturing conditions.
In the analyzed protein, multiple subunits were suspected due to the presence of bands in the SDS-PAGE, even without any reducing agents. Identifying subunits is vital as it informs us about a protein's inferred quaternary structure, which in the given scenario, was further unveiled when reducing agents were applied.

Disulfide Bonds

Disulfide bonds are covalent bonds that form between two cysteine residues within a protein, playing a crucial role in maintaining and stabilizing tertiary and quaternary structures. These bonds can be broken using reducing agents like dithiothreitol (DTT), which was seen in the exercise, where bands changed indicating the breaking of disulfide links.

• The transition in bands from 180 kDa to separate 90 kDa suggests the presence of a disulfide-linked dimer becoming monomers upon reduction.
• This change helps confirm the subunit composition and reveals the arrangement and linkage of protein subunits.

Understanding disulfide bonds is essential in biochemistry, allowing researchers to manipulate protein structures and map out assembly and interaction sites critical for their function.